The broad goal of the proposed research project is to improve treatment outcomes for patients with FLT3-mutant acute myeloid leukemia (AML). FLT3 mutations are detected in approximately 25-30% of AML patients. Recently, we have found that two drug-resistant FLT3 kinase domain mutations represent the most common cause of relapse in AML patients undergoing treatment with AC220 (quizartinib), the first clinically active investigational inhibitor of FLT3 for patients with AML. We have preliminary evidence that the most common AC220-resistant mutation in AML patients is sensitive in the laboratory to crenolanib, a drug that has already undergone clinical trial evaluation for other types of cancer. Crenolanib is also active in the laboratory against activation loop (AL) mutations in FLT3, which are found in 5-10% of AML patients. The second AC220-resistant mutation is sensitive to ponatinib, another investigational drug that is active in patients with chronic myeloid leukemia. The specific aims of this proposal are to: 1) assess the activity of crenolanib against clinically-important FLT3 mutant isoforms in vitro and in vivo; 2) assess the ability of crenolanib and ponatinib to collectively suppress all secondary FLT3-ITD kinase domain mutants in vitro and in vivo; and 3) determine the structural and functional impact of AL mutations in FLT3. This research focuses on AML, which impacts approximately 13,000 Americans annually, the majority of whom die of their disease within 1-2 years. It is anticipated that the proposed research will: 1) determine the potential of a novel FLT3 inhibitor as a treatment for AML associated with activation loop mutations in FLT3; 2) inform the development of a possible combination FLT3 inhibitor clinical trial; 3) provide insights into the structural and signaling impacts of activation loop mutations. The research design includes in vitro and in vivo as well as structural and signaling studies, and in addition, translational studies of primary samples, including from AML patients who have developed resistance to AC220 treatment.